Agrobacterium cells as microprojectile coating: a novel approach to enhance stable transformation rates in strawberry

Abstract

The effect of combining Agrobacterium tumefaciens infection and biolistic bombardment on the transformation of strawberry (Fragaria × ananassa Duch.) cv. Chandler, was evaluated. Bombarding leaf explants with uncoated gold particles followed by Agrobacterium infection did not improve transformation, and yielded similar percentages of shoot regeneration in the presence of kanamycin in bombarded and non-bombarded explants (7.2%). In a novel approximation, gold particles were coated with Agrobacterium cells and used to bombard leaf explants. Helium pressures of 4.5, 6.2 and 7.6 MPa and target distances of 3 and 9 cm were tested. An average of 96.2% of the explants showed β-glucuronidase (GUS) expression 15 d after bombardment, in comparison with 26.6% in explants bombarded with gold particles coated with the plasmid pGUSINT or 58.3% in non-bombarded Agrobacterium-infected explants. After 25 weeks of culture, the highest transformation frequency was obtained using a 6.2 MPa helium pressure and 3 cm target distance, yielding 69% kanamycin-resistant explants and a final transformation fre-quency of 20.7%. These values were 4.5 times higher for kanamycin-resistant explants (69% with biolistic vs 16% with Agrobacterium infection) and 2.9 times higher for transformation frequency (20.7 vs 7%,) compared with those obtained with standard Agrobacterium transformation procedures (Barceló et al. 1998, Plant Cell, Tiss. Org. Cult. 54, 29–36). More than 15 independent transgenic plants obtained by the Agrobacterium-coated particle system were acclimatized and confirmed as transgenics by GUS activity and PCR. Segregation analysis of kanamycin resistance has been performed in seven independent lines, three of which contained a single insertion of the T-DNA.

An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

“…Effective selection, using suitable selectable marker genes, can lead to a substantial reduction in the number of untransformed regenerants [42]. Most binary vectors used for transformation of strawberry carry the nptII gene conferring kanamycin resistance [10,33,44,45,46]. Based on the literature the possible doses of kanamycin that have been applied for selection of putative transformants from leaf explant of strawberry ranged from 25 mg/L [47,48] to 50 mg/L [33,49].…”

Molecular Characterization of a Stable Antisense Chalcone Synthase Phenotype in Strawberry (Fragaria × ananassa)

“…and wild strawberry ( F. vesca ) have already been reported. Although they are considered to be relatively amenable to transformation using Agrobacterium ( − ), transformation frequencies are greatly influenced by the cultivar and the procedure used ( , ). The technique is being used successfully to genetically engineer virus resistance ().…”

Nanotechnology Strategies for Plant Genetic Engineering

“…by combining Agrobacterium infection and particle bombardment. [ 56 ] They found that gold particles coated with Agrobacterium could achieve increased expression of β‐glucuronidase (GUS) within 15 days after particle bombardment, while uncoated gold particles did not improve GUS expression. Therefore, the combined use of Agrobacterium and particle bombardment can achieve higher transformation efficiency in certain plants.…”

“…by combining Agrobacterium infection and particle bombardment. [56] They found that gold particles coated with Agrobacterium could achieve increased expression of β-glucuronidase (GUS) within 15 days after particle Adv. Mater.…”